Reversible terminator nucleotides and methods of use

ABSTRACT

Disclosed herein a reversible terminator nucleotides and methods of use.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. application Ser.No. 12/493,125 filed Jun. 26, 2009 which is a continuation of U.S.patent application Ser. No. 11/763,405 filed Jun. 14, 2007 (now U.S.Pat. No. 7,554,949), which is a non-provisional application under 35U.S.C. §119(e) of provisional application No. 60/818,009 filed Jun. 30,2006, the disclosures of each of which is incorporated herein byreference.

INTRODUCTION

Methods of polynucleotide sequencing that employ reversible terminatornucleotides typically involve multiple steps to identify a single baseand to regenerate a 3′ terminus of the sequencing primer which isrequired permit identification of each succeeding base of thepolynucleotide. These steps typically include incorporation of areversible terminator at the 3′ terminus of the primer, washing away theunincorporated reversible terminators and other sequencing reagents,detection of the label attached to the incorporated reversibleterminator, removal of the label from the incorporated terminator, andremoval of blocking group from the reversible terminator. Therefore,current methods of polynucleotide sequencing using reversibleterminators are time-consuming, labor intensive, and have a high-costassociated with the identification of each base. Therefore, there is aneed in the art for reversible terminator nucleotides and methods of usethat reduce the number of steps required for the identification of eachbase of a polynucleotide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides the structure of an exemplary reversible terminatornucleotide comprising a dye label, a phosphate blocking group and afragmentable linker comprising a phosphate trigger moiety;

FIG. 2 provides an example of a fragmentable linker comprising afluorescent label and a phosphate trigger moiety; and

FIG. 3 provides an example of a fragmentable linker comprising afluorescent label and a phosphate trigger moiety.

DETAILED DESCRIPTION

This section should describe in detail the various different embodimentsof the invention(s). It should use headings to help organize the variouscomponent parts of the invention(s).

Disclosed herein are compositions and methods for sequencingpolynucleotides, including single-molecule sequencing. The compositionsdisclosed herein include reversible terminator nucleotides that can beincorporated at the 3′ terminus of a polynucleotide by a polymerase in atemplate dependent/directed manner. In some embodiments, the reversibleterminator nucleotides generally include a hydroxyl group at the3′-position of a sugar moiety, a blocking group at the 2′-position ofthe sugar moiety, and a label attached to the base. Upon incorporationof a reversible terminator nucleotide, the blocking group renders thepolynucleotide non-extendible by the polymerase. In some embodiments,the incorporated reversible terminators also are generally resistant to3′-5′ exonucleases or “proofreading activities” of polymerases.

Definitions

As used herein, the following terms are intended to have the followingmeanings:

“Nucleoside” refers to a compound consisting of a purine, deazapurine,or pyrimidine nucleoside base, e.g., adenine, guanine, cytosine, uracil,thymine, 7-deazaadenine, 7-deazaguanosine, that is linked to theanomeric carbon of a pentose sugar at the 1′ position, such as a ribose,2′-deoxyribose, or a 2′,3′-di-deoxyribose. When the nucleoside base ispurine or 7-deazapurine, the pentose is attached at the 9-position ofthe purine or deazapurine, and when the nucleoside base is pyrimidine,the pentose is attached at the 1-position of the pyrimidine (see, e.g.,Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman 1992)). The term“nucleotide” as used herein refers to a phosphate ester of a nucleoside,e.g., a mono-, a di-, or a triphosphate ester, wherein the most commonsite of esterification is the hydroxyl group attached to the C-5position of the pentose. “Nucleotide 5′-triphosphate” refers to anucleotide with a triphosphate. ester group at the 5′ position. The term“nucleoside/tide” as used herein refers to a set of compounds includingboth nucleosides and/or nucleotides.

“Nucleobase polymer or oligomer” refers to two or more nucleobasesconnected by linkages that permit the resultant nucleobase polymer oroligomer to hybridize to a polynucleotide having a complementarynucleobase sequence. Nucleobase polymers or oligomers include, but arenot limited to, poly- and oligonucleotides (e.g., DNA and RNA polymersand oligomers), poly- and oligonucleotide analogs and poly- andoligonucleotide mimics, such as polyamide or peptide nucleic acids.Nucleobase polymers or oligomers can vary in size from a fewnucleobases, from 2 to 40 nucleobases, to several hundred nucleobases,to several thousand nucleobases, or more.

“Polynucleotide or oligonucleotide” refers to nucleobase polymers oroligomers in which the nucleobases are connected by sugar phosphatelinkages (sugar-phosphate backbone). Exemplary poly- andoligonucleotides include polymers of 2′-deoxyribonucleotides (DNA) andpolymers of ribonucleotides (RNA). A polynucleotide may be composedentirely of ribonucleotides, entirely of 2′-deoxyribonucleotides orcombinations thereof.

In some embodiments, a nucleobase polymer is an polynucleotide analog oran oligonucleotide analog. By “polynucleotide analog or oligonucleotideanalog” is meant nucleobase polymers or oligomers in which thenucleobases are connected by a sugar phosphate backbone comprising oneor more sugar phosphate analogs. Typical sugar phosphate analogsinclude, but are not limited to, sugar alkylphosphonates, sugarphosphoramidites, sugar alkyl- or substituted alkylphosphotriesters,sugar phosphorothioates, sugar phosphorodithioates, sugar phosphates andsugar phosphate analogs in which the sugar is other than 2′-deoxyriboseor ribose, nucleobase polymers having positively charged sugar-guanidylinterlinkages such as those described in U.S. Pat. No. 6,013,785 andU.S. Pat. No. 5,696,253 (see also, Dagani, 1995, Chem. & Eng. News4-5:1153; Dempey et al., 1995, J. Am. Chem. Soc. 117:6140-6141). Suchpositively charged analogues in which the sugar is 2′-deoxyribose arereferred to as “DNGs,” whereas those in which the sugar is ribose arereferred to as “RNGs.” Specifically included within the definition ofpoly- and oligonucleotide analogs are locked nucleic acids (LNAs; see,e.g., Elayadi et al., 2002, Biochemistry 41:9973-9981; Koshkin et al.,1998, J. Am. Chem. Soc. 120:13252-3; Koshkin et al., 1998, TetrahedronLetters, 39:4381-4384; Jumar et al., 1998, Bioorganic & MedicinalChemistry Letters 8:2219-2222; Singh and Wengel, 1998, Chem. Commun.,12:1247-1248; WO 00/56746; WO 02/28875; and, WO 01/48190.

In some embodiments, a nucleobase polymer is a polynucleotide mimic oroligonucleotide mimic. “Polynucleotide mimic or oligonucleotide mimic”refers to a nucleobase polymer or oligomer in which one or more of thebackbone sugar-phosphate linkages is replaced with a sugar-phosphateanalog. Such mimics are capable of hybridizing to complementarypolynucleotides or oligonucleotides, or polynucleotide oroligonucleotide analogs or to other polynucleotide or oligonucleotidemimics, and may include backbones comprising one or more of thefollowing linkages: positively charged polyamide backbone withalkylamine side chains as described in U.S. Pat. Nos. 5,786,461,5,766,855, 5,719,262, 5,539,082 and WO 98/03542 (see also, Haaima etal., 1996, Angewandte Chemie Int'l Ed. in English 35:1939-1942; Lesnicket al., 1997, Nucleotid. 16:1775-1779; D'Costa et al., 1999, Org. Lett.1:1513-1516; Nielsen, 1999, Curr. Opin. Biotechnol. 10:71-75); unchargedpolyamide backbones as described in WO 92/20702 and U.S. Pat. No.5,539,082; uncharged morpholino-phosphoramidate backbones as describedin U.S. Pat. Nos. 5,698,685, 5,470,974, 5,378,841, and 5,185,144 (seealso, Wages et al., 1997, BioTechniques 23:1116-1121); peptide-basednucleic acid mimic backbones (see, e.g., U.S. Pat. No. 5,698,685);carbamate backbones (see, e.g, Stirchak and Summerton, 1987, J. Org.Chem. 52:4202); amide backbones (see, e.g., Lebreton, 1994, Synlett.February, 1994:137); methylhydroxyl amine backbones (see, e.g., Vasseuret al., 1992, J. Am. Chem. Soc. 114:4006); 3′-thioformacetal backbones(see, e.g., Jones et al., 1993, J. Org. Chem. 58:2983) and sulfamatebackbones (see, e.g., U.S. Pat. No. 5,470,967). All of the precedingreferences are herein incorporated by reference.

“Peptide nucleic acid” or “PNA” refers to poly- or oligonucleotidemimics in which the nucleobases are connected by amino linkages(uncharged polyamide backbone) such as described in any one or more ofU.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,718,262,5,736,336, 5,773,571, 5,766,855, 5,786,461, 5,837,459, 5,891,625,5,972,610, 5,986,053, 6,107,470, 6,451,968, 6,441,130, 6,414,112 and6,403,763; all of which are incorporated herein by reference. The term“peptide nucleic acid” or “PNA” shall also apply to any oligomer orpolymer comprising two or more subunits of those polynucleotide mimicsdescribed in the following publications: Lagriffoul et al., 1994,Bioorganic & Medicinal Chemistry Letters, 4:1081-1082; Petersen et al.,1996, Bioorganic & Medicinal Chemistry Letters, 6:793-796; Diderichsenet al., 1996, Tett. Lett. 37:475-478; Fujii et al., 1997, Bioorg. Med.Chem. Lett. 7:637-627; Jordan et al., 1997, Bioorg. Med. Chem. Lett.7:687-690; Krotz et al., 1995, Tett. Lett. 36:6941-6944; Lagriffoul etal., 1994, Bioorg. Med. Chem. Lett. 4:1081-1082; Diederichsen, 1997,Bioorg. Med. Chem. 25 Letters, 7:1743-1746; Lowe et al., 1997, J. Chem.Soc. Perkin Trans. 1, 1:539-546; Lowe et al., 1997, J. Chem. Soc. PerkinTrans. 11:547-554; Lowe et al., 1997, I. Chem. Soc. Perkin Trans. 11:555-560; Howarth et al., 1997, I. Org. Chem. 62:5441-5450; Altmann etal., 1997, Bioorg. Med. Chem. Lett., 7:11.19-1122; Diederichsen, 1998,Bioorg. Med. Chem. Lett., 8:165-168; Diederichsen et al., 1998, Angew.Chem. mt. Ed., 37:302-305; Cantin et al., 1997, Tett. Lett.,38:4211-4214; Ciapetti et al., 1997, Tetrahedron, 53:1167-1176;Lagriffoule et al., 1997, Chem. Eur. 1. 3:912-919; Kumar et al., 2001,Organic Letters 3(9):1269-1272; and the Peptide-Based Nucleic AcidMimics (PENAMs) of Shah et al. as disclosed in WO 96/04000.

Some examples of PNAs are those in which the nucleobases are attached toan N-(2-aminoethyl)-glycine backbone, i.e., a peptide-like, amide-linkedunit (see, e.g., U.S. Pat. No. 5,719,262; Buchardt et al., 1992, WO92/20702; Nielsen et al., 1991, Science 254:1497-1500).

In some embodiments, a nucleobase polymer is a chimeric oligonucleotide.By “chimeric oligonucleotide” is meant a nucleobase polymer or oligomercomprising a plurality of different polynucleotides, polynucleotideanalogs and polynucleotide mimics. For example a chimeric oligo maycomprise a sequence of DNA linked to a sequence of RNA. Other examplesof chimeric oligonucleotides include a sequence of DNA linked to asequence of PNA, and a sequence of RNA linked to a sequence of PNA.

In some embodiments, a nucleobase polymer is a chimeric oligonucleotide.By “chimeric oligonucleotide” is meant a nucleobase polymer or oligomercomprising a plurality of different polynucleotides, polynucleotideanalogs and polynucleotide mimics. For example a chimeric oligo maycomprise a sequence of DNA linked to a sequence of RNA. Other examplesof chimeric oligonucleotides include a sequence of DNA linked to asequence of PNA, and a sequence of RNA linked to a sequence of PNA.

In some embodiments, various components of the disclosed methods,including but not limited to primers, reversible terminator nucleotides,and other reaction compartments, can comprise a detectable moiety.“Detectable moiety,” “detection moiety” or “label” refer to a moietythat renders a molecule to which it is attached detectable oridentifiable using known detection systems (e.g., spectroscopic,radioactive, enzymatic, chemical, photochemical, biochemical,immunochemical, chromatographic, physical (e.g., sedimentation,centrifugation, density), electrophoretic, gravimetric, or magneticsystems). Non-limiting examples of labels include quantum dots, isotopiclabels (e.g., radioactive or heavy isotopes), magnetic labels; spinlabels, electric labels; thermal labels; colored labels (e.g.,chromophores), luminescent labels (e.g., fluorescers, chemiluminescers),enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase,luciferase, β-galactosidase) (Ichiki, et al., 1993, J. Immunol.150(12):5408-5417; Nolan, et al., 1988, Proc. Natl. Acad. Sci. USA85(8):2603-2607)), antibody labels, and chemically modifiable labels. Inaddition, in some embodiments, such labels include components ofligand-binding partner pairs (e.g., antigen-antibody (includingsingle-chain antibodies and antibody fragments, e.g., FAb, F(ab)′₂,Fab′, Fv, etc. (Fundamental Immunology 47-105 (William E. Paul ed.,5^(th) ed., Lippincott Williams & Wilkins 2003)), hormone-receptorbinding, neurotransmitter-receptor binding, polymerase-promoter binding,substrate-enzyme binding, inhibitor-enzyme binding (e.g.,sulforhodamine-valyl-alanyl-aspartyl-fluoromethylketone(SR-VAD-FMK-caspase(s) binding), allosteric effector-enzyme binding,biotin-streptavidin binding, digoxin-antidigoxin binding,carbohydrate-lectin binding, Annexin V-phosphatidylserine binding(Andree et al., 1990, J. Biol. Chem. 265(9):4923-8; van Heerde et al.,1995, Thromb. Haemost. 73(2):172-9; Tait et al., 1989, J. Biol. Chem.264(14):7944-9), nucleic acid annealing or hybridization, or a moleculethat donates or accepts a pair of electrons to form a coordinatecovalent bond with the central metal atom of a coordination complex. Invarious exemplary embodiments the dissociation constant of the bindingligand can be less than about 10⁻⁴-10⁻⁶M⁻¹, less than about 10⁻⁵ to 10⁻⁹M⁻¹, or less than about 10⁻⁷-10⁻⁹ M⁻¹.

“Fluorescent label,” “fluorescent moiety,” and “fluorophore” refer to amolecule that may be detected via its inherent fluorescent properties.Examples of suitable fluorescent labels include, but are not limited to,fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin,coumarin, methyl-coumarins, pyrene, Malachite Green, stilbene, LuciferYellow, Cascade BlueJ, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640,phycoerythrin, LC Red 705, Oregon green, Alexa-Fluor dyes (Alexa Fluor350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568,Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680),Cascade Blue, Cascade Yellow and R-phycoerythrin (PE), FITC, Rhodamine,Texas Red (Pierce, Rockford, Illinois), Cy5, Cy5.5, Cy7 (Amersham LifeScience, Pittsburgh, PA) and tandem conjugates, such as but not limitedto, Cy5PE, Cy5.5PE, Cy7PE, Cy5.5APC, Cy7APC. In some embodiments,suitable fluorescent labels also include, but are not limited to, greenfluorescent protein (GFP; Chalfie, et al., 1994, Science263(5148):802-805), EGFP (Clontech Laboratories, Inc., Palo Alto,Calif.), blue fluorescent protein (BFP; Quantum Biotechnologies, Inc.Montreal, Canada; Heim et al, 1996, Curr. Biol. 6:178-182; Stauber,1998, Biotechniques 24(3):462-471;), enhanced yellow fluorescent protein(EYFP; Clontech Laboratories, Inc., Palo Alto, Calif.), and renilla (WO92/15673; WO 95/07463; WO 98/14605; WO 98/26277; WO 99/49019; U.S. Pat.Nos. 5,292,658, 5,418,155, 5,683,888, 5,741,668, 5,777,079, 5,804,387,5,874,304, 5,876,995, and 5,925,558). Further examples of fluorescentlabels are found in Haugland, Handbook of Fluorescent Probes andResearch, 9^(th) Edition, Molecule Probes, Inc. Eugene, Oreg. (ISBN0-9710636-0-5).

By “microparticle”, “microsphere”, “microbead”, “bead” and grammaticalequivalents herein are meant a small discrete synthetic particle. Asknown in the art, the composition of beads can vary depending on thetype of assay in which they are used and, therefore, selecting amicrobead composition is within the abilities of the practitioner.Suitable bead compositions include those used in peptide, nucleic acidand organic synthesis, including, but not limited to, plastics,ceramics, glass, polystyrene, methylstyrene, acrylic polymers,paramagnetic materials (U.S. Pat. Nos. 4,358,388, 4,654,267, 4,774,265,5,320,944, 5,356,713), thoria sol, carbon graphite, titanium dioxide,latex or cross-linked dextrans such as Sepharose, agarose, cellulose,carboxymethyl cellulose, hydroxyethyl cellulose, proteinaceous polymer,nylon, globulin, DNA, cross-linked micelles and Teflon may all be used(see, e.g., Microsphere Detection Guide from Bangs Laboratories,Fishers, Ind.), Beads are also commercially available from, for example,Bio-Rad Laboratories (Richmond, Calif.), LKB (Sweden), Pharmacia(Piscataway, N.J.), IBF (France), Dynal Inc. (Great Neck, N.Y.). In someembodiments, beads may contain a cross-linking agent, such as, but notlimited to divinyl benzene, ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, N,N′methylene-bis-acrylamide, adipic acid,sebacic acid, succinic acid, citric acid, 1,2,3,4-butanetetracarboxylicacid, or 1,10 decanedicarboxylic acid or other functionally equivalentagents known in the art. In various exemplary embodiments, beads can bespherical, non-spherical, egg-shaped, irregularly shaped, and the like.The average diameter of a microparticle can be selected at thediscretion of the practitioner. However, generally the average diameterof microparticle can range from nanometers (e.g. about 100 nm) tomillimeters (e.g. about 1 mm) with beads from about 0.2 μm to about 200μm being preferred, and from about 0.5 to about 10 μm being particularlypreferred, although in some embodiments smaller or larger beads may beused, as described below.

In some embodiments a microparticle can be porous, thus increasing thesurface area available for attachment to another molecule, moiety, orcompound (e.g., a primer). Thus, microparticles may have additionalsurface functional groups to facilitate attachment and/or bonding. Thesegroups may include carboxylates, esters, alcohols, carbamides,aldehydes, amines, sulfur oxides, nitrogen oxides, or halides. Methodsof attaching another molecule or moiety to a bead are known in the art(see, e.g., U.S. Pat. Nos. 6,268,222, 6,649,414). In some embodiments, amicroparticle can further comprise a label.

An “extended polynucleotide” refers to a polynucleotide to which one ormore additional nucleotides have been added or otherwise incorporated(e.g., covalently bonded to).

A “template polynucleotide” refers to a polynucleotide to which a primercan hybridize and be extended. Accordingly, template polynucleotidesinclude subsequences that are at least partially complementary to aprimer. Template polynucleotides can be derived from essentially anysource. To illustrate, template nucleic acids are optionally derived orisolated from, e.g., cultured microorganisms, uncultured microorganisms,complex biological mixtures, tissues, sera, pooled sera or tissues,multispecies consortia, ancient, fossilized or other nonlivingbiological remains, environmental isolates, soils, groundwaters, wastefacilities, deep-sea environments, or the like. Further, templatepolynucleotides optionally include or are derived from, e.g., individualcDNA molecules, cloned sets of cDNAs, cDNA libraries, extracted RNAs,natural RNAs, in vitro transcribed RNAs, characterized oruncharacterized genomic DNAs, cloned genomic DNAs, genomic DNAlibraries, enzymatically fragmented DNAs or RNAs, chemically fragmentedDNAs or RNAs, physically fragmented DNAs or RNAs, or the like. Templatenucleic acids can also be chemically synthesized using techniques knownin the art. In addition, template nucleic acids optionally correspond toat least a portion of a gene or are complementary thereto. As usedherein, a “gene” refers to any segment of DNA associated with abiological function. Thus, genes include coding sequences andoptionally, the regulatory sequences required for their expression.Genes also optionally include non-expressed DNA segments that, forexample, form recognition sequences for other proteins.

Polynucleotides are “extended” or “elongated” when additionalnucleotides (or other analogous molecules, e.g., reversible terminatornucleotides) are incorporated into the nucleic acids. For example, apolynucleotide is optionally extended by a nucleotide incorporatingpolymerase that typically adds nucleotides at the 3′ terminal end of apolynucleotide.

An “extendible polynucleotide” refers to a nucleotide to which at leastone other nucleotide can be added or covalently bonded, e.g., in areaction catalyzed by a polymerase once the extendible polynucleotide isincorporated. An extendible polynucleotide is typically extended byadding another nucleotide at a 3′-position of the sugar moiety.

A “non-extendible” nucleotide refers to a nucleotide, which uponincorporation into a polynucleotide at least substantially inhibitsfurther extension of the polynucleotide.

“Parallel reaction” as used herein refers to a reaction comprising aplurality of discrete regions suitable for performing a plurality ofreactions simultaneously. Virtually any number of polynucleotides cananalyzed in parallel. For example, in various exemplary embodiments,hundreds, thousands, hundreds of thousands, millions, and even greaternumbers of polynucleotides can be analyzed in parallel by the disclosedmethods. In various exemplary embodiments the numbers of polynucleotidesanalyzed in parallel can be at least 2, 100, 500, 1000, 10000, 50000,100000, 300000, 500000, or 1000000, and even greater.

A “moiety” or “group” refers to one of the portions into whichsomething, such as a molecule, is divided (e.g., a functional group,substituent group, or the like). For example, a nucleotide typicallycomprises a basic group (e.g., adenine, thymine, cytosine, guanine,uracil, or an analog basic group), a sugar moiety (e.g., a moietycomprising a sugar ring or an analog thereof), and one or more phosphategroups.

A “heterocyclic ring” refers to a monocyclic or bicyclic ring that iseither saturated, unsaturated, or aromatic, and which comprises one ormore heteroatoms independently selected from nitrogen, oxygen andsulfur. A heterocyclic ring may be attached to the sugar moiety, oranalog thereof, of a nucleotide of the invention via any heteroatom orcarbon atom. Exemplary heterocyclic rings include morpholinyl,pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl,oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl,tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl,tetrahydrothiopyranyl, furyl, benzofuranyl, thiophenyl, benzothiophenyl,pyrrolyl, indolyl, isoindolyl, azaindolyl, pyridyl, quinolinyl,isoquinolinyl, oxazolyl, isooxazolyl, benzoxazolyl, pyrazolyl,imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isothiazolyl,pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl,phthalazinyl, quinazolinyl, and the like.

A “homocyclic ring” refers to a saturated or unsaturated (but notaromatic) carbocyclic ring, such as cyclopropane, cyclobutane,cyclopentane, cyclohexane, cycloheptane, cyclohexene, and the like.

An “alkyl group” refers to a linear, branched, or cyclic saturatedhydrocarbon moiety and includes all positional isomers, e.g., methyl,ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl,1,1-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl,2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1,1-dimethylpropyl,1,2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl,4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl,2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl,2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl,1-ethyl-1-methylpropyl and 1-ethyl-2-methylpropyl, n-hexyl,cyclohexyl,n-heptyl, n-octyl, 2-ethylhexyl, n-nonyl, n-decyl and the like. An alkylgroup typically comprises about 1-20 carbon atoms and more typicallycomprises about 2-15 carbon atoms. Alkyl groups can be substituted orunsubstituted.

An “alkenyl group” refers to a linear, branched, or cyclic unsaturatedhydrocarbon moiety that comprises one or more carbon-carbon doublebonds. Exemplary alkenyl groups include ethenyl, 2-propenyl, 2-butenyl,3-butenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl, 2-pentenyl,3-pentenyl, 4-pentenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl,3-methyl-2-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl,3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl, 1,2-dimethyl-2-propenyl,1-ethyl-2-propenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl,1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl,4-methyl-2-pentenyl, 1-methyl-3-pentenyl, 2-methyl-3-pentenyl,3-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl-4-pentenyl,2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl,1,1-dimethyl-2-butenyl, 1,1-dimethyl-3-butenyl, 1,2-dimethyl-2-butenyl,1,2-dimethyl-3-butenyl, 1,3-dimethyl-2-butenyl, 1,3-dimethyl-3-butenyl,2,2-dimethyl-3-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl,3,3-dimethyl-2-butenyl, 1-ethyl-2-butenyl, 1-ethyl-3-butenyl,2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1,1,2-trimethyl-2-propenyl,1-ethyl-1-methyl-2-propenyl, 1-ethyl-2-methyl-2-propenyl, and the like.An alkenyl group typically comprises about 1-20 carbon atoms and moretypically comprises about 2-15 carbon atoms. Alkenyl groups can besubstituted or unsubstituted.

An “alkynyl group” refers to a linear, branched, or cyclic unsaturatedhydrocarbon moiety that comprises one or more carbon-carbon triplebonds. Representative alkynyl groups include, e.g., 2-propynyl,2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 2-pentynyl, 3-pentynyl,4-pentynyl, 1-methyl-2-butynyl, 1-methyl-3-butynyl, 2-methyl-3-butynyl,1,1-dimethyl-2-propynyl, 1-ethyl-2-propynyl, 2-hexynyl, 3-hexynyl,4-hexynyl, 5-hexynyl, 1-methyl-2-pentynyl, 1-methyl-3-pentynyl,1-methyl-4-pentynyl, 2-methyl-3-pentynyl, 2-methyl-4-pentynyl,3-methyl-4-pentynyl, 4-methyl-2-pentynyl, 1,1-dimethyl-2-butynyl,1,1-dimethyl-3-butynyl, 1,2-dimethyl-3-butynyl, 2,2-dimethyl-3-butynyl,3,3-dimethyl-1-butynyl, 1-ethyl-2-butynyl, 1-ethyl-3-butynyl,2-ethyl-3-butynyl 1-ethyl-1-methyl-2-propynyl, and the like. An alkynylgroup typically comprises about 1-20 carbon atoms and more typicallycomprises about 2-15 carbon atoms. Alkynyl groups can be substituted orunsubstituted.

An “alkoxy group” refers to an alkyl group that comprises an oxygen atomand includes, e.g., methoxy, ethoxy, propoxy, butoxy, pentoxy,heptyloxy, octyloxy, and the like.

A “halo group” refers to a group that comprises a halogen atom, such asF, Cl, Br, or I.

An “aryl group” refers to a substituent group of atoms or moiety that isderived from an aromatic compound. Exemplary aryl groups include, e.g.,phenyl groups, benzyl groups, tolyl groups, xylyl groups, or the like.Aryl groups optionally include multiple aromatic rings (e.g., diphenylgroups, etc.). In addition, an aryl group can be substituted orunsubstituted.

An “aryloxy group” refers an aryl group that comprises an oxygen atomand includes, e.g., phenoxy, chlorophenoxy, methylphenoxy,methoxyphenoxy, butylphenoxy, pentylphenoxy, benzyloxy, and the like.

An “alkyl-aryl group” refers to a group that comprises alkyl and arylmoieties.

An “ether group” refers to a linear, branched, or cyclic moiety thatcomprises two carbon atoms attached to a single oxygen atom. Exemplaryether groups include, e.g., methoxymethyl, methoxyethyl, methoxypropyl,ethoxyethyl, and the like.

A “thioether group” refers to a linear, branched, or cyclic moiety thatcomprises two carbon atoms attached to a single sulfur atom andincludes, e.g., methylthiomethyl, methylthioethyl, methylthiopropyl, andthe like.

An “alkylamine group” refers to an amino group that comprises at leastone alkyl group.

An “alkenylamine group” refers to an amino group that comprises at leastone alkenyl group.

An “alkynylamine group” refers to an amino group that comprises at leastone alkynyl group.

An “ester group” refers to a class of organic compounds that includesthe general formula RCOOR′, where R and R′ are independently selectedfrom an alkyl group, an alkenyl group, an alkynyl group, an aryl group,or combinations thereof.

A “polyaminoacid” refers to compound or group that comprises two or moreamino acid residues. Exemplary polyaminoacids include peptides,polypeptides, proteins, and the like.

An “aldehyde group” refers to an organic group that includes the formulaCHO.

An “alcohol group” refers to an organic group that includes at least onehydroxy group.

A “silyl group” refers to a class of compounds that includes the generalformula SiRR′R″, where R, R′, and R″ are independently an H, an alkylgroup, an alkenyl group, an alkynyl group, an aryl group, or acombination of such groups.

A “phosphate analog” refers to a moiety that can be cleaved by aphosphatase, such as, an alkaline phosphatase.

A “sequence” of a polynucleotide refers to the order and identity ofnucleotides in the nucleic acid. A sequence is typically read in the 5′to 3′ direction.

“Attached” refers to interactions including, but not limited to,covalent bonding, ionic bonding, chemisorption, physisorption, andcombinations thereof.

A “linker” or “spacer” refers to a chemical moiety that covalently ornon-covalently (e.g., ionically, etc.) attaches a compound orsubstituent group to, e.g., a solid support, another compound or group,or the like. For example, a linker optionally attaches a label (e.g., afluorescent dye, a radioisotope, etc.) to a 2′-terminator nucleotide orthe like. Linkers are typically bifunctional chemical moieties and incertain embodiments, they comprise cleavable attachments, which can becleaved by, e.g., heat, an enzyme, a chemical agent, electromagneticradiation, etc. to release materials or compounds from, e.g., a solidsupport, another compound, etc. A careful choice of linker allowscleavage to be performed under appropriate conditions compatible withthe stability of the compound and assay method. Generally a linker hasno specific biological activity other than to, e.g., join chemicalspecies together or to preserve some minimum distance or other spatialrelationship between such species. However, the constituents of a linkermay be selected to influence some property of the linked chemicalspecies such as three-dimensional conformation, net charge,hydrophobicity, etc. Additional description of linker molecules isprovided in, e.g., Lyttle et al. (1996) Nucleic Acids Res. 24(14):2793,Shchepino et al. (2001) Nucleosides, Nucleotides, & Nucleic Acids20:369, Doronina et al (2001) Nucleosides, Nucleotides, & Nucleic Acids20:1007, Trawick et al. (2001) Bioconjugate Chem. 12:900, Olejnik et al.(1998) Methods in Enzymology 291:135, Pljevaljcic et al. (2003) J. Am.Chem. Soc. 125(12):3486, Ward, et. al., U.S. Pat. No. 4,711,955,Stavrianopoulos, U.S. Pat. No. 4,707,352, and Stavrianopoulos, U.S. Pat.No. 4,707,440, which are each incorporated by reference.

A “modified” enzyme refers to an enzyme comprising a sequence in whichat least one amino acid differs from a reference sequence, such as anative or wild-type form of the enzyme or another modified form of theenzyme, e.g., when the two sequences are aligned for maximum identity.Exemplary modifications include monomer insertions, deletions, andsubstitutions. The modified enzymes (e.g., polymerases) can be createdby various diversity generating methods. Although essentially any methodcan be used to produce a modified enzyme, certain exemplary techniquesinclude recombining (e.g., via recursive recombination, syntheticrecombination, or the like) two or more nucleic acids encoding one ormore parental enzymes, or by mutating one or more nucleic acids thatencode enzymes, e.g., using recursive ensemble mutagenesis, cassettemutagenesis, random mutagenesis, in vivo mutagenesis, site directedmutagenesis, or the like. A nucleic acid encoding a parental enzymetypically includes a gene that, through the mechanisms of transcriptionand translation, produces an amino acid sequence corresponding to aparental enzyme, e.g., a native form of the enzyme. Modified enzymesalso include chimeric enzymes that have identifiable component sequences(e.g., structural and/or functional domains, etc.) derived from two ormore parents. Also included within the definition of modified enzymesare those comprising chemical modifications (e.g., attached substituentgroups, altered substituent groups, etc.) relative to a referencesequence.

A “solid support” refers to a solid material which can be derivatizedwith, or otherwise attached to, a chemical moiety, such as apolynucleotide or the like. Exemplary solid supports include a plate, abead, a microbead, a fiber, a whisker, a comb, a hybridization chip, amembrane, a single crystal, a ceramic layer, a a slide, aself-assembling monolayer, and the like.

“Cleavage” refers to a process of releasing a material or compound ormoiety from another material or compound or moiety.

Detailed Description

It is to be understood that both the foregoing general description,including the drawings, and the following detailed description areexemplary and explanatory only and are not restrictive of thisdisclosure. In this disclosure, the use of the singular includes theplural unless specifically stated otherwise. Also, the use of “or” means“and/or” unless stated otherwise. Similarly, “comprise,” “comprises,”“comprising” “include,” “includes,” and “including” are not intended tobe limiting.

Disclosed herein are compositions and methods for sequencingpolynucleotides, including single-molecule sequencing. The compositionsdisclosed herein include reversible terminator nucleotides that can beincorporated at the 3′ terminus of a polynucleotide by a polymerase in atemplate dependent/directed manner. In some embodiments, the reversibleterminator nucleotides generally include a hydroxyl group at the3′-position of a sugar moiety, a blocking group at the 2′-position ofthe sugar moiety, and a label attached to the base. Upon incorporationof a reversible terminator nucleotide, the blocking group renders thepolynucleotide non-extendible by the polymerase. In some embodiments,the incorporated reversible terminators also are generally resistant to3′-5′ exonucleases or “proofreading activities” of polymerases.

Following incorporation, the label of the reversible terminatornucleotide can detected to identify the base of the terminatornucleotide and therefore the corresponding base of the templatepolynucleotide. To permit a further round of extension, thepolynucleotide can be treated with one or more reagents under conditionsthat remove the blocking group and the label. Therefore, the blockinggroup and the label can be removed under identical conditions. Uponremoval of the label and blocking group, the 3′ terminus of thepolynucleotide can be further extended in a template dependent manner.

The structure of an exemplary reversible terminator nucleotide is shownin Formula I:

in which

-   -   R₁ represents H, OH, a hydrophilic group, or a hydrophobic        group;    -   B represents a nucleobase comprising at least one homocyclic        ring, at least one heterocyclic ring (with or without exocyclic        heteroatoms), or at least one aryl group, or combinations        thereof;    -   BG represents a reversible blocking group;    -   Z represents O, CH₂, or S    -   represents a single or double bond.

In some embodiments, a reversible terminator nucleotide can include atleast 1, 2, or 3 phosphate groups and/or phosphate analogs attached atthe 5′ position. In some embodiments, a reversible terminator nucleotidecomprises a label. In some embodiments, R₁ and BG may be at the 2′ and3′ positions, respectively.

Non-limiting examples of B groups of formula I include adenine,cytosine, guanine, thymine, uracil, 5-propynyl-uracil,2-thio-5-propynyl-uracil, 5-methylcytosine, pseudoisocytosine,2-thiouracil and 2-thiothymine, 2-aminopurine,N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine,N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine),N8-(7-deaza-8-aza-adenine), isoC and isoG. Other non-limiting examplesof suitable B groups include nucleobases disclosed in U.S. Pat. No.5,432,272, 6,001,983, 6,037,120, 61,040,496, 6,357,163, 6,617,106,6,977,161; U.S. Patent Publication Nos. 20040106108, 20050037398,20060078936; EP1358352, EP1590482; and W09220702, WO9220703, WO0233126and WO04065550. Thus, the skilled artisan will appreciate that a B groupof Formula I can be any naturally occurring or synthetic nucleobase thatis suitable for hybridizing to a polynucleotide in a sequence-specificmanner.

Blocking groups (BG) can be any moiety suitable for substantiallyinhibiting extension of a polynucleotide that comprises a 3′ reversibleterminator nucleotide of Formula I. The mechanism by which a BG inhibitsextension can be by various methods, including but not limited to charge(e.g., positive or negative charge) or steric inhibition. In someembodiments, a BG can optionally render a polynucleotide substantiallyresistant to 3′-5′ exonucleases and/or the “proofreading activities” ofpolymerases. In addition to substantially inhibiting extension, the BGof Formula I are reversible. By “reversible” herein is meant that the BGcan be substantially removed to yield a 3′ terminus suitable for primerextension in a template directed manner. The removal of a BG can beaccomplished using various methods and reagents, including but notlimited to, an enzyme, such as an alkaline phosphatase.

In some embodiments, a BG can be SO₃.

In some embodiments, a BG can be C(O)R₉, wherein R₉ represents H, analkyl group, a benzyl group, an aryl group, an alkenyl group, an alkynylgroup, or combinations thereof

In some embodiments, a BG can comprise the Formula II:

wherein

-   -   where X represents O, S, NR₃, CR₃R₄, or SiR₃R₄;    -   Y represents CR₅R₆R₇, SiR₅R₆R₇, OR₅, SR₅, or NHR₅;    -   R₂ represents H, OH, NHR₈, SR₈, an alkyl group, a benzyl group,        an aryl group, an alkenyl group, an alkynyl group, an alkoxy        group, or combinations thereof; and    -   R₃, R₄, R₅, R₆, R₇, and R₈ are independently selected from H, an        alkyl group, a benzyl group, an aryl group, an alkenyl group, an        alkynyl group, or combinations thereof.

In some embodiments, a BG can comprise the Formula III:

wherein

-   -   X represents CR₃R₄R₅, SiR₃R₄R₅, OR₃, SR₃, or NHR₃;    -   R₂ represents H, NHR₆, SR₆, an alkyl group, a benzyl group, an        aryl group, an alkenyl group, an alkynyl group, an alkoxy group,        or combinations thereof; and    -   R₃, R₄, R₅, and R₆ are independently selected from H, an alkyl        group, a benzyl group, an aryl group, an alkenyl group, an        alkynyl group, or combinations thereof.

In some embodiments, a BG can be a phosphate (—PO₄).

In some embodiments, a BG can be a SO₃.

In some embodiments, a BG can be C(O)R.

Methods of synthesizing the BG groups and their equivalents are known inthe art.

The reversible terminators disclosed herein optionally comprise at leastone label. In some embodiments, a label can be appended to thenucleobase (B) of a reversible terminator, for example, at homocyclicring, a heterocyclic ring, or an aryl group (e.g., via C-5 of apyrimidine, N-4 of cytidine, N-7 of a purine, N-6 of adenosine, C-8 of apurine, or another attachment site as in the art). In some embodiments,a label can be attached via an amide, an ester, a thioester, an ether, athioether, a carbon-carbon, or other type of covalent bond. In someembodiments, a label can be attached to a sugar moiety (e.g., a ribosesugar) or an analog thereof of a reversible terminator nucleotide. Insome embodiments, a label can be attached to a BG, such as, the BG ofFormulas II and III or a phosphate, such as, by an amide, an ester, athioester, an ether, a thioether, a carbon-carbon, or other bond. Insome embodiments, a label can be appended to a reversible terminatornucleotide via a linker as described herein.

In various exemplary embodiments, a label comprises a fluorescent dye(e.g., a rhodamine dye (e.g., R6G, R110, TAMRA, ROX, etc.), afluorescein dye (e.g., JOE, VIC, TET, HEX, FAM, etc.), a halofluoresceindye, a cyanine dye (e.g., CY3, CY3.5, CY5, CY5.5, etc.), a BODIPY® dye(e.g., FL, 530/550, TR, TMR, etc.), an ALEXA FLUOR® dye (e.g., 488, 532,546, 568, 594, 555, 653, 647, 660, 680, etc.), a dichlororhodamine dye,an energy transfer dye (e.g., BIGDYE™ v 1 dyes, BIGDYE™ v 2 dyes,BIGDYE™ v 3 dyes, etc.), Lucifer dyes (e.g., Lucifer yellow, etc.),CASCADE BLUE®, Oregon Green, and the like. Other exemplary dyes areprovided in Haugland, Molecular Probes Handbook of Fluorescent Probesand Research Products, Ninth Ed. (2003) and the updates thereto.Fluorescent dyes are generally readily available from various commercialsuppliers including, e.g., Molecular Probes, Inc. (Eugene, Oreg.),Amersham Biosciences Corp. (Piscataway, N.J.), Applera Corp./AppliedBiosystems Group (Foster City, Calif.). Other exemplay labels include,e.g., biotin, weakly fluorescent labels (Yin et al. (2003) Appl EnvironMicrobiol. 69(7):3938; Babendure et al. (2003) Anal. Biochem. 317(1):1;and Jankowiak et al. (2003) Chem Res Toxicol. 16(3):304),non-fluorescent labels, colorimetric labels, chemiluminescent labels(Wilson et al. (2003) Analyst. 128(5):480; Roda et al. (2003)Luminescence 18(2):72), Raman labels, electrochemical labels,bioluminescent labels (Kitayama et al. (2003) Photochem Photobiol.77(3):333; Arakawa et al. (2003) Anal. Biochem. 314(2):206; and Maeda(2003) J. Pharm. Biomed. Anal. 30(6):1725), and an alpha-methyl-PEGlabeling reagent as described in, e.g., U.S. Provisional PatentApplication No. 60/428,484, filed on Nov. 22, 2002.

In some embodiments, a label comprises a radioisotope, such as 3H, ¹⁴C,²²Na, ³²P, ³³P, ³⁵S, ⁴²K, ⁴⁵Ca, ⁵⁹Fe, ¹²⁵I, ²⁰³Hg, and the like. In someembodiments, a label can comprise at least one mass-modifying group,including but not limited to, deuterium, F, Cl, Br, I, S, N₃, XY, CH₃,SPO₄, BH₃, SiY₃, Si(CH₃)₃, Si(CH₃)₂(C₂H₅), Si(CH₃)(C₂H₅)₂, Si(C₂H₅)₃,(CH₂)_(n)CH₃, (CH₂)_(n)NY₂, CH₂CONY₂, (CH₂)_(n)OH, CH₂F, CHF₂, CF₃, anda phosphorothioate group, where X is O, NH, NY, S, NHC(S),OCO(CH)_(n)COO, NHCO(CH₂)_(n)COO, OSO₂2O, OCO(CH₂)_(n), NHC(S)NH,OCO(CH₂)_(n)S, OCO(CH₂)S, NC₄4O₂H₂S, OPO(O-alkyl), or OP(O-alkyl);wherein n is an integer from 1 to 20 inclusive; and, Y is H, deuterium,an alkyl group, an alkoxy group, an aryl group, a polyoxymethylenegroup, a monoalkylated polyoxymethylene group, a polyethylene iminegroup, a polyamide group, a polyester group, a alkylated silyl group, aheterooligo, a polyaminoacid, a heterooligo/polyaminoacid group, or apolyethylene glycol group.

A large variety of linkers can be used to optionally link labels and BGto the reversible terminator nucleotides and will be apparent to one ofskill in the art. A linker is generally of a structure that issterically and electronically suitable for incorporation into apolynucleotide. In some embodiments, linkers can include an ether, athioether, a carboxamide, a sulfonamide, a urea, a urethane, ahydrazine, or other moieties. In some embodiments, a linker can comprisefrom about 1 and to about 25 non-hydrogen atoms selected from, e.g., C,N, O, P, Si, S, etc., and comprise essentially any combination ofexemplary groups, such as, an ether, a thioether, an amine, an ester, acarboxamide, a sulfonamide, hydrazide bonds, aromatic or heteroaromaticbonds. In some embodiments, a linker comprises a combination of singlecarbon-carbon bonds and carboxamide or thioether bonds. In someembodiments, longer linear segments of linkers can be utilized, thelongest linear segment typically contains between about three to about15 nonhydrogen atoms, including one or more heteroatoms.

Non-limiting examples of linker moieties include substituted (e.g.,functionalized) or unsubstituted groups, such as imidazole/biotinlinkers, polymethylene groups, arylene groups, alkylarylene groups,arylenealkyl groups, arylthio groups, amido alkyl groups, alkynyl alkylgroups, alkenyl alkyl groups, alkyl groups, alkoxyl groups, thio groups,amino alkyl groups, morpholine derivatized phosphates, peptide nucleicacids (e.g., N-(2-aminoethyl)glycine, etc.), and the like. (see, e.g.,U.S. Pat. Nos. 4,711,958, 5,047,519, 5,175,269, 5,328,824, 6,339,392;and U.S. Patent Publication No. 20020151711. Additional examples oflabeling and linkers are provided in Hermanson, Bioconjugate Techniques,Elsevier Science (1996).

In some embodiments, suitable linkers comprise photocleavable moieties,such as 2-nitrobenzyl moieties, alpha-substituted 2-nitrobenzyl moieties(e.g., 1-(2-nitrophenyl)ethyl moieties), 3,5-dimethoxybenzyl moieties,thiohydroxamic acid, 7-nitroindoline moieties, 9-phenylxanthyl moieties,benzoin moieties, hydroxyphenacyl moieties, NHS-ASA moieties, and thelike. Exemplary photocleavable linkers are described in U.S. PatentPublication No. 20030099972. In some embodiments, linkers includemetals, such as platinum atoms. (see, e.g., U.S. Pat. No. 5,714,327). Anumber of linkers of varying lengths are commercially available fromvarious suppliers including, e.g., Qiagen/Operon Technologies, Inc.(Alameda, Calif.), BD Biosciences Clontech (Palo Alto, Calif.), andMolecular BioSciences (Boulder, Colo.).

In some embodiments a suitable linker can be a fragmentable linker. By“fragmentable linker” as used herein refers to a linker that is capableof fragmenting in an electronic cascade self-elimination reaction. Invarious exemplary embodiments, fragmentation of the linker can result inthe removal of a label and/or a BG from the reversible terminatornucleotides, disclosed herein. Examples of fragmentable linkers aredisclosed in U.S. Patent Publication No. 20060003383.

In some embodiments, a fragmentable linker comprises a linker moiety anda trigger moiety. The linker moiety, the trigger moiety, and theoptional label and/or BG can be connected to the linker moiety in anyway that permits them to perform their respective functions. The triggermoiety comprises a substrate that can be cleaved or “activated” by aspecified trigger agent. Activation of the trigger moiety initiates aspontaneous rearrangement that results in the fragmentation of thelinker and release of the label and/or BG. In some embodiments, therelease of the label and/or BG from the reversible terminator nucleotidemay be caused by a ring closure mechanism. In some embodiments, a linkerfragments via an elimination reaction. Various elimination reactions,including but not limited to, 1,4-, 1,6- and 1,8-elimination reactionshave been used in the design of prodrugs and can be adapted for use inthe compositions and methods described herein. (see, e.g., WO02083180,Gopin et al. Angew Chem int Ed. 32:327-332 (2003), Niculescu-Duvaz etal. J Med Chem. 41:5297-5309 (1998), Florent et al. J Med Chem.41:3572-3581 (1998), Niculescu-Duvaz et al. J Med Chem. 42:2485-2489(1999), Greenwald et al. J Med Chem. 42:3657-3667 (1999), de Groot etal. Bioorg Med Chem Lett. 12:2371-2376 (2002), Ghosh et al. TetrahedronLetters 41:4871-4874 (2000), Dubowchik et al. Bioconjugate Chem.13:855-869 (2002), Michel et al. Atta-ur-Rahman (ed.) 21:157-180 (2000),Dinaut et al. Chem Commun. 1386-1387 (2001), Ohwada et al. Bioorg MedChem Lett. 12:2775-2780 (2002), de Groot et al. J Org Chem. 66:8815-8830(2001), Leu et al. J Med Chem. 42:3623-3628 (1999), Sauerbrei et al.Angew Chem Int Ed. 37:1143-1146 (1998), Veinberg et al. Bioorg Med ChemLett. 14:1007-1010 (2004), Greenwald et al. Bioconjugate Chem.14:395-403 (2003), and Lee et al. Angew Chem Int Ed. 43:1675-1678(2004).

Any means of activating the trigger moiety may be used, provided thatthe means used to activate the trigger moiety is capable of producing adetectable change, such as, a decrease in fluorescence and/or theremoval of a BG and/or primer extension. Selection of a particular meansof activation, and hence trigger moiety, may depend, in part, on theparticular fragmentation reaction, as well as on other factors.

In some embodiments, activation is based upon cleavage of the triggermoiety. In these embodiments, the trigger moiety comprises a cleavagesite that is cleavable by a chemical reagent or cleaving enzyme. Innon-limiting exemplary embodiments, a trigger moiety can comprise acleavage site that is cleavable by a sulfatase (e.g., SO₃ and analogsthereof), an esterase (e.g., C(O)R₉ and analogs thereof), or aphosphatase (e.g., PO₄ and analogs thereof. (Parent et al. CurrentOpinion in Genetics & Development 1997; 7(3):386-391; Ellis et al.Applied & Environmental Microbiology 2002; 68(1):31-36; Bogaerts et al.Environmental Toxicology & Chemistry 1998; 17(8):1600-1605; Choudhury.Journal of Histochemistry and Cytochemistry 1972; 20(7):507-517). As aspecific example, the trigger moiety can comprise a cleavage site thatis cleavable by a phosphatase, such as, alkaline phosphatase.

The trigger moiety can further comprise additional residues and/orfeatures that facilitate the specificity, affinity and/or kinetics ofthe cleaving enzyme. Depending upon the requirements of the particularcleaving enzyme, such cleaving enzyme “recognition moieties” cancomprise the cleavage site or, alternatively, the cleavage site may beexternal to the recognition moiety.

The chemical composition of the trigger moiety will depend upon, amongother factors, the requirements of the cleaving enzyme. For example, ifthe cleaving enzyme is a phosphatase, the trigger moiety can comprise aphosphate or a phosphate analog recognized and cleaved by the particularphosphatase. If the cleaving enzyme is a nuclease, the trigger moietycan comprise an oligonucleotide (or analog thereof) recognized andcleaved by a particular nuclease. If the cleaving enzyme is glycosidase,the trigger moiety can comprise a carbohydrate recognized and cleaved bya particular glycosidase (see, e.g., Florent et al. J Med Chem.41:3572-3581 (1998), Ghosh et al. Tetrahedron Letters 41:4871-4874(2000), Michel et al. Atta-ur-Rahman (ed.) 21:157-180 (2000), and Leu etal. J Med Chem. 42:3623-3628 (1999)). Exemplary structures recognizedand cleaved by lipases and esterases, such as phosphatases, are alsowell known and include structures disclosed in Ohwada et al. Bioorg MedChem Lett. 12:2775-2780 (2002), Sauerbrei et al. Angew Chem Int Ed.37:1143-1146 (1998), Greenwald et al. J Med Chem Lett. 43:475-487(2000), Dillon et al. Bioorg Med Chem Lett. 14:1653-1656 (1996), andGreenwald et al. J Med Chem. 47:726-734 (2004)). Exemplary structuresrecognized and cleaved by proteases/proteolytic enzymes are disclosed inNiculescu-Duvaz et al. J Med Chem. 41:5297-5309 (1998), Niculescu-Duvazet al. J. Med Chem. 42:2485-2489 (1999), Greenwald et al. J Med Chem.42:3657-36670 (1999), de Groot et al. Bioorg Med Chem Lett. 12:2371-2376(2002), Dubowchik et al. Bioconjugate Chem. 13:855-869 (2002), and deGroot et al. J Org Chem. 66:8815-8830 (2001). Exemplary structuresrecognized and cleaved by catalytic antibodies are disclosed in Gopin etal. Angew Chem Int Ed. 42:327-332 (2003), Dinaut et al. Chem Commun.1386-1387 (2001).

The linker moiety, the trigger moiety and the optional label and BG areconnected to the linker moiety in any way that permits them to performtheir respective functions. In some embodiments, the trigger moiety,label and/or BG are each, independently of the other, directly connectedto the linker moiety. In other embodiments, the trigger moiety, labeland/or BG can be, independently of the other, indirectly connected tothe linker moiety via one or more optional linkages. The optionallinkages can comprise a leaving group, which upon fragmentation of thesubstrate compound is released from the backbone of the linker, alongwith the moiety that is attached to it. For example, in someembodiments, the label can be attached to the backbone of the linkermoiety via a linkage comprising a leaving group, while BG can beattached to the backbone of the linker moiety via a stable linkage,e.g., a linkage that does not dissociate from the backbone of the linkerfollowing the fragmentation reaction.

In some embodiments, the trigger moiety also serves as the linkermoiety. In these embodiments, cleavage of the trigger moiety by aspecified trigger agent also results in fragmentation of the linker andrelease of the label and/or BG.

In some embodiments, the trigger moiety also serves as the BG. In theseembodiments cleavage of the trigger moiety by a specified trigger agentresults in fragmentation of the linker and removal of the BG. Therefore,in these embodiments, linker fragmentation and removal of the BG canoccur under identical conditions in a single step. As a result, a labelattached to the fragmentable linker and the BG can be removed underidentical conditions in a single step. An example of a trigger moietythat can also serve as a BG include but are not limited to a —PO₄, SO₃,and C(O)R₉.

An example of a reversible terminator nucleotide comprising a phosphateBG and trigger moiety is shown in FIG. 1, in which a fragmentable linkeris appended to a cytosine base. The phosphate on the fragmentable linkerserves as the trigger moiety. Cleavage of the phosphate trigger moietycauses fragmentation of the linker and removal of the dye from thecytosine base. Cleavage of the phosphate BG at the 2′ position of theribose sugar permits the addition of an additional nucleotide at the 3′position. Other examples of fragmentable linkers comprising phosphatetrigger moieties are shown in FIG. 2 and FIG. 3, in which thefragmentable linker comprises a fluorescent dyes. Methods of makingthese and other exemplary fragmentable linkers are disclosed in U.S.Patent Publication No. 20060003383.

In some embodiments, a phosphate BG and trigger moiety can be cleaved byan alkaline phosphatase (“ALP”) (orthophosphate monoesterphosphohydrolases [alkaline optimum]; (EC 3.1.3.1). In general, ALPs arehomodimeric nonspecific metalloenzymes that catalyze phosphomonoesterasereactions. (Coleman. Annu Rev Biophys Biomol Struct. 1992; 21:441-483;Holtz et al. FEBS Lett. 1999; 462:7-11; Holtz et al. J Biol Chem. 1999;274:8351-8354; Kim et al. Clin Chim Acta 1989; 186; 175-188; Manes etal. Genomics 1990; 8:541-554; McComb et al. Alkaline Phosphatase 1979Plenum Press, NY; Trowsdale et al. Biochem Soc Trans. 1990; 18:178-180).The reaction conditions for ALPS are well-known in the art andnon-limiting examples of ALPs suitable for use in the disclosed methodsinclude shrimp alkaline phosphatase (de Backer et al. J Mol Biol. 2002May 17; 318(5):1265-74; Olsen et al. Comp Biochem Physiol. 199199B:755-61), bovine intestinal alkaline phosphatase (Weissig et al.Biochem J. 1993; 290:503-508), and ALPs isolated from Pyrococcus abyssi(Erauso et al. Arch Microbiol. 1993; 160:338-349; Zappa et al. ApplEnviron Microbiol. 2001 October; 67(10):4504-11), Pyrococcus horikoshiisp. (Gonzalez et al. Extremophiles 1998; 2:123-130), Thermatoganeopolitana (Dong et al. Enzyme Microb Technol. 1997; 21:335-340),Thermus caldophilus GK24 (Park et al. FEMS Microbiol Lett. 1999;180:133-139), Thermus thermophilus (Pantazaki et al. Appl. BiochemBiotechnol. 1998; 75:249-259), Haloarcula (Goldman et al. J Bacteriol.1990; 172:7065-7070), Halobacterium (Bonet et al. Biochem Mol Biol Int.1994; 34:1109-1120; Fitt et al. Biochem J. 1979; 181:347-353),Prevotello intermedia (Ansai et al. FEBS Lett. 1998; 428:157-160),Bacillus (Hulett. Mol Microbiol. 1996; 19:933-939; Mori et al.Biotechnol Appl Biochem 1999; 29:235-239; Sharipova et al. Biochem.(Moscow) 1998; 63:1178-1182; Sharipova et al. Biochem Mol Biol Int.1996; 38:753-761; Spencer et al. J Bacteriol. 1981; 145:926-933), Vibrio(Hauksson et al. Enzyme Microb Technol. 2000; 27:66-73), Escherichiacoli (Coleman. Annu Rev Biophys Biomol Struct. 1992; 21:441-83; Dermanet al. J Bacteriol. 1991 December; 173(23):7719-22; Hulett et al. J BiolChem. 1991; 266:1077-1084; Janeway et al. Biochemistry 1993;32:1601-1609; Kane. Curr Opin Biotechnol. 1995; 6:494-500; Karamyshev etal. J Mol Biol. 1998 Apr. 10; 277(4):859-70; Kim et al. Biotechnol Lett.1998; 20:207-210; Murphy et al. J Mol Biol. 1995; 253:604-617; Murphy etal. J Biol Chem. 1993; 268:21497-21500), Bacteroides (Yamashita et al.Infect Immun. 1990; 58:2882-2887), Gadus morhua (Asgeirsson et al. CompBiochem Physiol. 1995; 110B:315-329), Scylla serrata (J Biochem CellBiol. 2000; 32:879-885), humans (Berget et al. PNAS USA 1987;84:695-698; Weiss et al. PNAS USA 1986; 83:7182-7186), insects (Eguchi.Comp Biochem Physiol. 1995; 111B:151-162), Neurospora (Morales et al.Braz J Med Biol Res. 2000; 33:905-912), Antarctic strain TABS (Rina etal. Eur J Biochem 2000; 267:1230-1238).

The reversible terminator nucleotides can be employed in virtually anymethod of analyzing or detecting a polynucleotide. In variousnon-limiting examples, suitable polynucleotide may be single ordouble-stranded, or a combination thereof, linear or circular, achromosome or a gene or a portion or fragment thereof, a regulatorypolynucleotide, a restriction fragment from, for example, a plasmid orchromosomal DNA, genomic DNA, mitochondrial DNA, DNA from a construct orlibrary of constructs (e.g., from a YAC, BAC or PAC library), RNA (e.g.,mRNA, rRNA or vRNA) or a cDNA or a cDNA library. As known in the art, acDNA is a single- or double-stranded DNA produced by reversetranscription of an RNA template. Therefore, some embodiments include areverse transcriptase and one or more primers suitable for reversetranscribing an RNA template into a cDNA. Reactions, reagents andconditions for carrying out such “RT” reactions are known in the art(see, e.g., Blain et al., 1993, J. Biol. Chem. 5:23585-23592; Blain etal., 1995, J. Virol. 69:4440-4452; PCR Essential Techniques 61-63,80-81, (Burke, ed., J. Wiley & Sons 1996); Gubler et al., 1983, Gene25:263-269; Gubler, 1987, Methods Enzymol., 152:330-335; Sellner et al.,1994, J. Virol. Method. 49:47-58; Okayama et al., 1982, Mol. Cell. Biol.2:161-170; and U.S. Pat. Nos. 5,310,652, 5,322,770, 6,300,073, thesedisclosures of which are incorporated herein by reference. In someembodiments, a polynucleotide may include a single polynucleotide (e.g.,a chromosome, plasmid) from which one or more different sequences ofinterest may clonally amplified and analyzed. Methods of clonalamplification are disclosed in *U.S. application Ser. No. 11/377,763.

In some embodiments, polynucleotides can be analyzed in a parallelmanner. For example, discrete or isolated polynucleotides can beanalyzed in parallel as a result of their attachment to a discrete areaof a surface or isolation in a well of a multi-well plate. Therefore, insome embodiments, at least at least 100, 500, 1000, 10000, 50000,100000, 300000, 500000, or 1000000 polynucleotides can be analyzed inparallel. Generally, parallel reactions produce discrete detectablesignals that can be associated or linked to individual polynucleotides.

In some embodiments, polynucleotides can be sequenced using thereversible terminator nucleotides based on sequencing-by-synthesistechniques. The sequencing-by-synthesis technique employs controlledsynthesis of nucleic acids (e.g., primer extension) and the detection ofeach incorporated reversible terminator nucleotide in a step-by-stepmanner. For example, in some embodiments, a primer can be hybridized toa polynucleotide that is immobilized to surface in the presence of fourreversible terminator nucleotide triphosphates each labeled with aspectrally resolvable fluorescent dye and a polymerase. Theincorporation of a first reversible terminator at the 3′ terminus of thesequencing primer prevents further extension. To detect the incorporatedreversible terminator, the unincorporated reversible terminators arewashed away and the incorporated reversible terminator is detected byits fluorescent label. To permit a further cycle of extension anddetection, the label and BG are removed from the incorporated reversibleterminator. In embodiments, that employ a phosphate BG and afragmentable linker comprising a phosphate trigger moiety to areversible terminator, the BG and label can be removed under identicalconditions using an alkaline phosphatase, such as shrimp alkalinephosphatase, as described above. Therefore, an enzymatic reaction to canbe employed to efficiency remove the label and BG under identicalconditions. Following removal of the label and BG from the incorporatedreversible terminator, the alkaline phosphatase can be inactivated orwashed away prior to the reintroduction of the four reversibleterminator nucleotide triphosphates and polymerase. For example, in someembodiments, shrimp alkaline phosphatase can be inactivated byincubation at 65° C. for 15 min.

In practicing the disclosed methods, many conventional techniques inmolecular biology and recombinant DNA can be used. These techniques arewell known in the art and disclosed in, for example, Current Protocolsin Molecular Biology, Volumes I, II, and III, 1997 (F. M. Ausubel ed.);Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, ThirdEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger),DNA Cloning: A Practical Approach, Volumes I and II, 1985 (D. N. Glovered.); Oligonucleotide Synthesis, 1984 (M. L. Gait ed.); Nucleic AcidHybridization, 1985, (Hames and Higgins); Transcription and Translation,1984 (Hames and Higgins eds.); Animal Cell Culture, 1986 (R. I. Freshneyed.); Immobilized Cells and Enzymes, 1986 (IRL Press); Perbal, 1984, APractical Guide to Molecular Cloning; the series, Methods in Enzymology(Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells, 1987(J. H. Miller and M. P. Calos eds., Cold Spring Harbor Laboratory); andMethods in Enzymology Vol. 154 and Vol. 155 (Wu and Grossman, and Wu,eds., respectively).

Methods of attaching a polynucleotide to either covalently ornon-covalently to a solid support or a surface are also known in theart. Solid supports can comprise various materials, including but notlimited to any one or more of plastic, glass, ceramic, metal, resin, geland a membrane. Exemplary types of solid supports include but are notlimited plates, slides, beads, microbeads, whiskers, fibers, combs,hybridization chips, membranes, single crystals, ceramics, andself-assembling monolayers.

Polynucleotides can be attached to a solid support by covalent bindingsuch as by conjugation with a coupling agent or by non-covalent bindingsuch as electrostatic interactions, hydrogen bonds or antibody-antigencoupling, or by combinations thereof. Exemplary coupling agents includebiotin/avidin, biotin/streptavidin, Staphylococcus aureus protein A/IgGantibody F_(c) fragment, and streptavidin/protein A chimeras (Sano etal. (1991) Bio/Technology 9:1378, which is incorporated by reference),or derivatives or combinations of these agents. Nucleic acids may beattached to the solid support by a photocleavable bond, an electrostaticbond, a disulfide bond, a peptide bond, a diester bond or a combinationof these bonds. Nucleic acids are also optionally attached to solidsupports by a selectively releasable bond such as 4,4′-dimethoxytritylor its derivative. Derivatives which have been found to be usefulinclude 3 or 4 [bis-(4-methoxyphenyl)]-methyl-benzoic acid,N-succinimidyl-3 or 4 [bis-(4-methoxyphenyl)]-methyl-benzoic acid,N-succinimidyl-3 or 4 [bis-(4-methoxyphenyl)]-hydroxymethyl-benzoicacid, N-succinimidyl-3 or 4 [bis-(4-methoxyphenyl)]-chloromethyl-benzoicacid, and salts of these acids. In some embodiments, polynucleotides canbe attached to solid supports via spacer moieties between the nucleicacids and the solid support.

In some embodiments, a polynucleotide can be attached to a solid supportvia a cleavable attachment. Cleavable attachments can be created byattaching cleavable chemical moieties between the polynucleotides andthe solid support including, e.g., an oligopeptide, oligonucleotide,oligopolyamide, oligoacrylamide, oligoethylene glycerol, alkyl chains ofbetween about 6 to 20 carbon atoms, and combinations thereof. Thesemoieties may be cleaved with, e.g., added chemical agents,electromagnetic radiation, or enzymes. Exemplary attachments cleavableby enzymes include peptide bonds which can be cleaved by proteases andphosphodiester bonds which can be cleaved by nucleases.

Chemical agents such as β-mercaptoethanol, dithiothreitol (DTT) andother reducing agents cleave disulfide bonds. Other agents that may beuseful include oxidizing agents, hydrating agents and other selectivelyactive compounds. Electromagnetic radiation such as ultraviolet,infrared and visible light cleave photocleavable bonds. Attachments mayalso be reversible, e.g., using heat or enzymatic treatment, orreversible chemical or magnetic attachments. Release and reattachmentcan be performed using, e.g., magnetic or electrical fields.

A variety of nucleic acid polymerases may be used in the sequencing andother methods described herein. In various exemplary embodiments, apolymerase can be a thermostable polymerase or a thermally degradablepolymerase. Suitable thermostable polymerases include, but are notlimited to, polymerases isolated from Thermus antranikianii, Thermusaquaticus, Thermus caldophilus, Thermus chliarophilus, Thermusfiliformis, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermusoshimai, Thermus ruber, Thermus rubens, Thermus scotoductus, Thermussilvanus, Thermus species Z05, Thermus species sps 17, Thermusthermophilus, Thermotoga maritima, Thermotoga neapolitana, Thermosiphoafricanus, Anaerocellum thermophilum, Bacillus caldotenax, Bacillusstearothermophilus, Pyrococcus woesei, Pyrococcus furiosus, andThermococcus litoralis. For example, the polymerase optionally lacks anF to Y mutation in helix O of the enzyme or otherwise lacks a mutationthat enhances incorporation of 3′-deoxynucleotides by the enzyme. Insome embodiments, a polymerase comprises a 3′-5′exonuclease activity. Insome embodiments, a polymerase can be thermodegradable, such as, E. coliDNA polymerase I, the Klenow fragment of E. coli DNA polymerase I, T4DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, and others.

Non-limiting examples of commercially available polymerases thatsuitable for the methods described herein include, but are not limitedto, TaqFS®, AmpliTaq CS (Perkin-Elmer), AmpliTaq FS (Perkin-Elmer),Kentaql (AB Peptide, St. Louis, Mo.), Taquenase (ScienTech Corp., St.Louis, Mo.), ThermoSequenase (Amersham), Bst polymerase, Vent_(R)(exo⁻)DNA polymerase, Reader™Taq DNA polymerase, VENT™ DNA polymerase (NewEngland Biolabs), DEEPVENT™ DNA polymerase (New England Biolabs),PFUTurbo™ DNA polymerase (Stratagene), Tth DNA polymerase, KlenTaq-1polymerase, SEQUENASE™ 1.0 DNA polymerase (Amersham Biosciences), andSEQUENASE 2.0 DNA polymerase (United States Biochemicals).

In some embodiments, a polymerase can be modified, which can enhance theincorporation of the reversible terminator nucleotides disclosed herein.Exemplary modified polymerases are disclosed in U.S. Pat. Nos.4,889,818, 5,374,553, 5,420,029, 5,455,170, 5,466,591, 5,618,711,5,624,833, 5,674,738, 5,789,224, 5,795,762, 5,939,292; and U.S. PatentPublication Nos. 20020012970, 20040005599. A non-limiting example of amodified polymerase includes G46E E678G CS5 DNA polymerase, G46E E678GCS5 DNA polymerase, E615G Taq DNA polymerase, ΔZO5R polymerase, and G46EL329A E678G CS5 DNA polymerase disclosed in U.S. Patent Publication No.20050037398. The production of modified polymerases can be accomplishedusing many conventional techniques in molecular biology and recombinantDNA described herein and known in the art. In some embodiments, whereinthe 2′-phosphate unblocking generates a 2′-hydroxyl, i.e., aribonucleotide, polymerase mutants, such as those described in U.S. Pat.No. 5,939,292, which incorporate NTPs as well as dNTPs can be used.

The products of methods disclosed herein can be analyzed by a widevariety of methods which can be selected at the discretion of thepractitioner. Analytical methods include but are not limited to gelelectrophoresis, capillary electrophoresis (CE: e.g., 3730 DNA Analyzer,3730x1 DNA Analyzer, 3100-Avant genetic analyser, and 270A-HT CapillaryElectrophoresis system (Applied Biosystems, Foster City, Calif.)) (see,e.g., U.S. Pat. Nos. RE37941, 5,384,024, 6,372,106, 6,372,484,6,387,234, 6,387,236, 6,402,918, 6,402,919, 6,432,651, 6,462,816,6,475,361, 6,476,118, 6,485,626, 6,531,041, 6,544,396, 6,576,105,6,592,733, 6,596,140, 6,613,212, 6,635,164, and 6,706,162) using variouspolymers (e.g., separation polymer (e.g., POP-4™ POP-6™, or POP-7™(Applied Biosystems, Foster City, Calif.), linear polyacrylamide (LPA:Kleparnik et al., 2001, Electrophoresis 22(4):783-8; Kotler et al.,2002, Electrophoresis 23(17):3062-70; Manabe et al., 1998,Electrophoresis 19:2308-2316)), chromatography, thin layerchromatography, or paper chromatography, by laser-induced fluorescence(see, e.g., U.S. Pat. Nos. 5,945,526, 5,863,727, 5,821,058, 5,800,996,5,332,666, 5,633,129, and 6,395,486), autoradiagraphy,chemiluminescence, mass spectrometric methods (see, e.g., U.S. PatentNos. 6,225,450 and 510,412), microcapillary electrophoretic methods(see, e.g., Doherty et al., 2004, Analytical Chemistry 76:5249-5256;Ertl et al., 2004, Analytical Chemistry 76:3749-3755; Haab et al., 1999,Analytical Chemistry 71:5137-5145 (1999); Kheterpal et al., 1999,Analytical Chemistry 71:31A-37A; Lagally et al., 2000, Sensors andActuators B 63:138-146; Lagally et al., 2001, Anal. Chem. 73:565-570;Lagally et al., 2003, Genetic Analysis Using a Portable PCR-CEMicrosystem, in Micro Total Analysis Systems Vol. 2, Northrup et al.(eds.) pp. 1283-1286; Liu et al., 1999, Anal. Chem. 71:566-573; Medintzet al., 2000, Electrophoresis 21:2352-2358; Medintz et al., 2001, GenomeResearch 11:413-421; Paegel et al., Current Opinions in Biotechnology14:42-50; Scherer et al., 1999, Electrophoresis 20:1508-1517; Shi etal., 1999; Analytical Chemistry 71:5354-5361; Wedemayer et al., 2001,BioTechniques 30:122-128; U.S. Pat. Nos. 6,787,015, 6,787,016; U.S.Application Nos. 20020166768, 20020192719, 20020029968, 20030036080,20030087300, 20030104466, 20040045827, 20040096960; EP1305615; and WO02/08744).

In some embodiments, the detectable signals generated by the labelsdisclosed herein can be detected using photomultiplier tubes (PMTs),charge-coupled devices (CCDs), intensified CCDs, photodiodes, avalanchephotodiodes, optical sensors, scanning detectors, or the like. Detectorssuch as these are readily available from various commercial sourcesincluding, e.g., Applied Biosystems (Foster City, Calif.). Detectionsystems of use in practicing the methods of the invention are describedfurther in, e.g., Skoog et al., Principles of Instrumental Analysis, 5thEd., Harcourt Brace College Publishers (1998) and Currell, AnalyticalInstrumentation: Performance Characteristics and Quality, John Wiley &Sons, Inc. (2000).

The primers (e.g., sequencing primers) employed in the methods disclosedherein, generally, should be sufficiently long to primetemplate-directed synthesis under the conditions of the reaction. Theexact lengths of the primers may depend on many factors, including butnot limited to, the desired hybridization temperature between theprimers and polynucleotides, the complexity of the different targetpolynucleotide sequences, the salt concentration, ionic strength, pH andother buffer conditions, and the sequences of the primers andpolynucleotides. The ability to select lengths and sequences of primerssuitable for particular applications is within the capabilities ofordinarily skilled artisans (see, e.g., Sambrook et al. MolecularCloning: A Laboratory Manual 9.50-9.51, 11.46; 11.50 (2d. ed., ColdSpring Harbor Laboratory Press); Sambrook et al., Molecular Cloning: ALaboratory Manual 10.1-10.10 (3d. ed. Cold Spring Harbor LaboratoryPress)). In some embodiments, the primers contain from about 15 to about35 nucleotides that are suitable for hybridizing to a targetpolynucleotide and form a substrate suitable for DNA synthesis, althoughthe primers may contain more or fewer nucleotides. Shorter primersgenerally require lower temperatures to form sufficiently stable hybridcomplexes with target sequences. The capability of polynucleotides toanneal can be determined by the melting temperature (“T_(m)”) of thehybrid complex. T_(m) is the temperature at which 50% of apolynucleotide strand and its perfect complement form a double-strandedpolynucleotide. Therefore, the T_(m) for a selected polynucleotidevaries with factors that influence or affect hybridization. In someembodiments, in which thermocycling occurs, the primers can be designedto have a melting temperature (“T_(m)”) in the range of about 60-75° C.Melting temperatures in this range tend to insure that the primersremain annealed or hybridized to the target polynucleotide at theinitiation of primer extension. The actual temperature used for a primerextension reaction may depend upon, among other factors, for example,the concentration of the primers. For reactions carried out with athermostable polymerase such as Taq DNA polymerase, in exemplaryembodiments primers can be designed to have a T_(m) in the range ofabout 60 to about 78° C. or from about 55 to about 70° C. The meltingtemperatures of the different primers can be different; however, in analternative embodiment they should all be approximately the same, i.e.,the T_(m) of each primer, for example, in a parallel reaction can bewithin a range of about 5° C. or less. The T_(m)s of various primers canbe determined empirically utilizing melting techniques that arewell-known in the art (see, e.g., Sambrook et al. Molecular Cloning: ALaboratory Manual 11.55-11.57 (2d. ed., Cold Spring Harbor LaboratoryPress)). Alternatively, the T_(m) of a primer can be calculated.Numerous references and aids for calculating T_(m)s of primers areavailable in the art and include, by way of example and not limitation,Baldino et al. Methods Enzymology. 168:761-777; Bolton et al., 1962,Proc. Natl. Acad. Sci. USA 48:1390; Bresslauer et al., 1986, Proc. Natl.Acad. Sci. USA 83:8893-8897; Freier et al., 1986, Proc. Natl. Acad. Sci.USA 83:9373-9377; Kierzek et al., Biochemistry 25:7840-7846; Montpetitet al., 1992, J. Virol. Methods 36:119-128; Osborne, 1991, CABIOS 8:83;Rychlik et al., 1990, Nucleic Acids Res. 18:6409-6412 (erratum, 1991,Nucleic Acids Res. 19:698); Rychlik. J. NIH Res. 6:78; Sambrook et al.Molecular Cloning: A Laboratory Manual 9.50-9.51, 11.46-11.49 (2d. ed.,Cold Spring Harbor Laboratory Press); Sambrook et al., MolecularCloning: A Laboratory Manual 10.1-10.10 (3d. ed. Cold Spring HarborLaboratory Press)); SantaLucia, 1998, Proc. Natl. Acad. Sci. USA95:1460-1465; Suggs et al., 1981, In Developmental Biology UsingPurified Genes (Brown et al., eds.), pp. 683-693, Academic Press;Wetmur, 1991, Crit. Rev. Biochem. Mol. Biol. 26:227-259, whichdisclosures are incorporated by reference. Any of these methods can beused to determine a T_(m) of a primer.

As the skilled artisan will appreciate, in general, the relativestability and therefore, the T_(m)s, of RNA:RNA, RNA:DNA, and DNA:DNAhybrids having identical sequences for each strand may differ. Ingeneral, RNA:RNA hybrids are the most stable (highest relative T_(m))and DNA:DNA hybrids are the least stable (lowest relative T_(m)).Accordingly, in some embodiments, another factor to consider, inaddition to those described above, when designing a primer is thestructure of the primer and target polynucleotide. For example, in theembodiment in which an RNA polynucleotide is reverse transcribed toproduce a cDNA, the determination of the suitability of a DNA primer forthe reverse transcription reaction should include the effect of the RNApolynucleotide on the T_(m) of the primer. Although the T_(m)s ofvarious hybrids may be determined empirically, as described above,examples of methods of calculating the T_(m) of various hybrids arefound at Sambrook et al. Molecular Cloning: A Laboratory Manual 9.51(2d. ed., Cold Spring Harbor Laboratory Press).

The sequences of primers useful for the disclosed methods are designedto be substantially complementary to regions of the targetpolynucleotides. By “substantially complementary” herein is meant thatthe sequences of the primers include enough complementarity to hybridizeto the target polynucleotides at the concentration and under thetemperature and conditions employed in the reaction and to be extendedby the DNA polymerase.

The compositions and reagents described herein can be packaged intokits. In some embodiments, a kit comprises one or more reversibleterminator-nucleotides. In some embodiments, each reversible nucleotidecan comprise a label (e.g., a fluorescent dye, a radioisotope, amass-modifying group etc.). In some embodiments, a label can be attachedto a reversible terminator nucleotide by a fragmentable linker. In someembodiments, the BG of a reversible terminator nucleotide and thetrigger moiety can be removed under identical conditions by the actionof an enzyme, such as, an alkaline phosphatase. Therefore, in someembodiments, a kit can include a trigger agent suitable for activating atrigger moiety and removing the BG from a reversible terminatornucleotides. In some embodiments, a kit can comprise a polymerase,including but not limited to, a modified polymerase as described herein.In some embodiments, a kit can comprise one or more sequencing primers.In some embodiments, a kit can further include a template polynucleotidesuitable for sequencing which can optionally be attached to a surface orsolid support. In some embodiments, a kit can comprise one or morereaction compartments comprising reagents suitable for performing areaction, such as a sequencing reaction, selected at the discretion of apractitioner. For example, in some embodiments, a kit can comprise oneor more reaction compartments comprising one more sequencing reagents.In some embodiments, a component of a kit can be used in conjunctionwith one or more reagents from commercially available kits, including,but not limited to, those available from Applied Biosystems (i.e., BigDye® Terminator Cycle Sequencing Kit), Epicentre (i.e., SequiTherm™Cycle Sequencing Kit), Amersham (i.e., DYEnamic Direct Dye-Primer CycleSequencing Kits), Boehringer Mannheim (i.e., CycleReader™ DNA SequencingKit), Bionexus Inc. (i.e., AccuPower DNA Sequencing Kit), and USB cyclesequencing kits (i.e., Thermo Sequenase™ Cycle Sequencing Kit).

The various components included in the kit are typically contained inseparate containers, however, in some embodiments, one or more of thecomponents can be present in the same container. Additionally, kits cancomprise any combination of the compositions and reagents describedherein. In some embodiments, kits can comprise additional reagents thatmay be necessary or optional for performing the disclosed methods. Suchreagents include, but are not limited to, buffers, molecular sizestandards, control polynucleotides, and the like.

In this application, the use of the singular includes the plural unlessspecifically stated otherwise. The section headings used herein are fororganizational purposes only and are not to be construed as limiting thesubject matter described in any way. While the present teachings aredescribed in conjunction with various embodiments, it is not intendedthat the present teachings be limited to such embodiments. On thecontrary, the present teachings encompass various alternatives,modifications, and equivalents, as will be appreciated by those of skillin the art.

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

1-29. (canceled)
 30. A polynucleotide in which the 3′ terminalnucleotide comprises a base and a sugar in which a label is attached tosaid base and a protecting group is attached to said sugar, wherein saidlabel and said protecting group can be detached from said polynucleotideby an enzyme.
 31. The polynucleotide of claim 30, wherein said label andsaid protecting group can be detached from said polynucleotide underidentical conditions.
 32. The polynucleotide of claim 30, wherein saidlabel and said protecting group comprise a phosphate moiety.
 33. Thepolynucleotide of claim 30, wherein said label and said protecting groupcomprise a SO₃ moiety.
 34. The polynucleotide of claim 30, wherein saidlabel and said protecting group comprise a C(O)R moiety.
 35. Thepolynucleotide of claim 30, wherein said enzyme is a phosphatase. 36.The polynucleotide of claim 35, wherein said phosphatase is shrimpalkaline phosphatase.
 37. The polynucleotide of claim 30, wherein saidenzyme is a sulfatase.
 38. The polynucleotide of claim 30, wherein saidenzyme is an esterase.
 39. The polynucleotide of claim 30, wherein saidlabel is a fluorescent label.
 40. The polynucleotide of claim 30,wherein said label is attached to said base by a fragmentable linker.41. The polynucleotide of claim 40, wherein said fragmentable linkercomprises a trigger moiety selected from a group consisting of PO₄, SO₃,and C(O)R.
 42. The polynucleotide of claim 41, wherein said triggermoiety is a substrate for said enzyme. 43-51. (canceled)